This invention relates to in vivo methods for detecting protein interactions and isolating novel proteins.
Many cell cycle regulatory proteins have been identified using yeast two-hybrid systems like the interaction trap (Gyuris et al., Cell 75:791, 1993; Harper et al., Cell 75:805, 1993; Serrano et al., Nature 366:704, 1993; Hannon et al., Genes & Dev. 7:2378, 1993). In two-hybrid systems (Fields and Song, Nature 340:245, 1989), two proteins are expressed in yeast: one contains a DNA-binding moiety (the "bait", the other a transcription activation domain. If the proteins interact, they activate transcription of a reporter gene that contains a binding site for the DNA-binding protein. Typically, the interaction trap (Gyuris et al., supra) uses E. coli LexA repressor as the DNA-binding moiety and two different reporter genes, LEU2 and lacz, that each contain upstream LexA operators. Proteins that may interact with the bait, such as those encoded by members of cDNA libraries, are fused to an activation domain and expressed conditionally under the control of the yeast GAL1 promoter. To conduct an interactor hunt, cells that contain a bait are transformed with a library plasmid that expresses activation-tagged cDNA proteins, and transformants that contain proteins that associate with the bait are selected because they grow in the absence of leucine and form blue colonies on X-Gal medium. The most sensitive LEU2 reporter allows detection of interacting proteins with estimated K.sub.d s less than 10.sup.-6 M (Gyuris et al., supra). Interacting proteins specific for the bait are identified as those that do not interact with unrelated baits.